Characterization of molecules in nanofluidics

ABSTRACT

Methods are provided for detecting and quantitating molecules using fluidics. In preferred embodiments, the methods comprise analyzing blood to detect the presence of circulating DNA or cells from a fetus or tumor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/795,847, filed on Oct. 27, 2017, which is a continuation of U.S.application Ser. No. 14/768,422, having a 371(c) date of Aug. 17, 2015and now U.S. Pat. No. 9,809,855, which is the national phase ofInternational Application PCT/US2014/017226 filed Feb. 19, 2014, whichclaims the benefit of U.S. Provisional Application No. 61/767,219, filedFeb. 20, 2013, each of which is hereby incorporated by reference in itsentirety.

SUMMARY

According to some embodiments herein, a method of characterizing asample is provided. The method can comprise labeling a plurality ofsample molecules with at least a first label, wherein the samplemolecules comprise polynucleotide sequences of a first genomic fragmentor fragments of interest, and wherein first genomic fragment orfragments of interest correspond to a possibly abnormal genomic regionof the sample. The method can comprise providing a plurality of labeledreference molecules, wherein the reference molecules comprisepolynucleotide sequences of a reference genomic fragment or fragments,and wherein the reference genomic fragment or fragments are known not tocorrespond to the possibly abnormal genomic region. As used herein“correspond to a possibly abnormal genomic region” and variations ofthis root term includes genomic fragments that overlap with or areencompassed by an abnormal chromosomal region, including, but notlimited to a duplication, deletion, inversion, translocation, and oraneuploid chromosome or fragment thereof. As such, a genomic fragment orfragment can correspond to an abnormal genomic region that is eitherpresent (e.g. a duplication) or absent (e.g. a deletion, for example ifthe genomic fragment would be encompassed by or overlap with the deletedregion). The method can comprise translocating the plurality of labeledsample molecules and the plurality of labeled reference molecules thougha fluidic channel. The method can comprise detecting signals from thelabeled sample molecules so as to ascertain at least a first pattern orplurality of patterns characteristic of the first genomic fragment orfragments of interest; and a second pattern or plurality of patternscharacteristic of the reference genomic fragment or fragments. Themethod can comprise correlating signals ascertaining the first patternor plurality of patterns to signals ascertaining the second pattern orplurality of patterns. In some embodiments, labeling comprises labelingthe sample molecules with a first label, and wherein the referencemolecules comprise a second label, in which the first label isconfigured to produce the first pattern or plurality of patterns, and inwhich the second label is configured to produce the second pattern orplurality of patterns, and in which the first label and the second labelare different from each other. In some embodiments, labeling compriseslabeling with a first label, in which the first pattern or plurality ofpatterns and the second pattern or plurality of patterns each comprisethe first label, and in which the first pattern or plurality of patternsand second pattern or plurality of patterns are different from eachother. In some embodiments, the method further comprised labeling thereference molecules so as to produce the labeled reference molecules,wherein the labeled reference molecules comprise the second pattern orplurality of patterns. In some embodiments, the labeled referencemolecules and sample molecules are from the sample. In some embodiments,the labeled reference molecules are from a different tissue of the sameorganism as the sample. In some embodiments, the labeled referencemolecules and sample molecules are from different organisms. In someembodiments, the signal from the labeled reference molecules comprisesan electronically or optically stored value or set of values. In someembodiments, the method further comprises labeling a second plurality ofsample molecules from a second sample with at least the first label,wherein the second plurality of sample molecules comprise polynucleotidesequences of the first genomic fragment or fragments of interest,wherein the second plurality of sample molecules is known to notcorrespond to the chromosomal abnormality, and translocating the secondplurality of sample molecules through the fluidic channel, and detectingsignals from the labeled sample molecules so as to ascertain at leastthe first pattern or plurality of patterns characteristic of the firstgenomic fragment or fragments of interest; and the second pattern orplurality of patterns characteristic of the reference genomic fragmentor fragments. In some embodiments, the method further comprises aligningthe positions of the patterns to positions of patterns in a referencegenome. In some embodiments, the sample molecules are from a samplecomprising the possible genomic abnormality, and the reference genomicfragment or fragments comprise the first chromosome or fragment thereof,in which the reference genomic fragments are from a second sample knownto not comprise the genomic abnormality. In some embodiments, the firstgenomic fragment or fragments of interest comprise a sex chromosome or aleast one fragment thereof, and the reference genomic fragment orfragments comprise an autosome or at least one fragment thereof. In someembodiments, the first genomic fragment or fragments of interestcomprise a first autosome or at least one fragment thereof, selectedfrom the group consisting of: human chromosome 21, human chromosome 13,human chromosome 14, human chromosome 15, human chromosome 16, humanchromosome 18, and human chromosome 22, and fragments thereof, and thereference genomic fragment or fragments comprise a second autosome or atleast one fragment thereof, wherein the second autosome or fragmentthereof is different than the first autosome or fragment thereof. Insome embodiments, correlating signals comprises using the ratio (K)between the signal arising from a plurality of labeled sample moleculesor portions thereof (S1, S2 . . . Sn) and the signal arising from thereference (C):K1=S1/C, K2=S2/C . . . Kn=Sn/C. In some embodiments, thefirst label comprises at least one of a fluorescent label, a radioactivelabel, a magnetic label, or a non-optical label. In some embodiments,the second label comprises at least one of a fluorescent label, aradioactive label, a magnetic label, or a non-optical label. In someembodiments, labeling comprises nicking one strand of a double-strandedDNA at a first sequence motif with a nicking endonuclease; and labelingthe DNA. In some embodiments, the method further comprises repairing atleast some of the nicks on the DNA. In some embodiments, the nicks arenot repaired. In some embodiments, the label comprises a transcriptionalterminator. In some embodiments, labeling with the first label comprisestagging at least one sequence motif of the sample molecules with a DNAbinding entity selected from the group consisting of: a non-cuttingrestriction enzyme, a zinc finger protein, an antibody, a transcriptionfactor, a transcription activator like domain, a DNA binding protein, apolyamide, a triple helix forming oligonucleotide, and a peptide nucleicacid, and a methyltransferase. In some embodiments, labeling with thefirst label comprises tagging at least one sequence motif of the samplemolecules with a methyltransferase. In some embodiments, the methodfurther comprises labeling the sample molecule with anon-sequence-specific label. The non-sequence specific label can bedifferent from the first label and the second label. In someembodiments, the possible abnormal genomic region comprises at least oneof a translocation, addition, amplification, transversion, inversion,aneuploidy, polyploidy, monosomy, trisomy, trisomy 21, trisomy 13,trisomy 14, trisomy 15, trisomy 16, trisomy 18, trisomy 22, triploidytetraploidy, or sex chromosome aneuploidy. In some embodiments, thegenetic abnormality comprises at least one of a trisomy or monosomy.

According to some embodiments herein, a method of characterizing asample is provided. The method can comprise labeling a plurality ofsequence-specific locations on a polynucleotide sequence of a samplemolecule. The method can comprise linearizing at least a portion of thesample molecule in a fluidic channel. The method can comprisequantifying a signal from the labels on the sample molecule. The methodcan comprise comparing a quantity of the signal from the sample moleculeto a quantity of signal from a reference molecule. The method cancomprise determining a presence or absence of a genetic abnormality inthe sample DNA when the quantity of the signal from the sample moleculediffers from the quantity of the signal arising from the referencemolecule. In some embodiments, the sample molecule and the referencemolecule are from the same organism. In some embodiments, the samplemolecule and the reference molecule are from different tissues of thesame organism. In some embodiments, the sample molecule and thereference molecule are from different organisms. In some embodiments,the signal from the quantity of signal from the reference moleculecomprises an electronically or optically stored value or set of values.In some embodiments, the sample molecule comprises a DNA. In someembodiments, the genetic abnormality comprises at least one of atranslocation, addition, amplification, transversion, inversion,aneuploidy, polyploidy, monosomy, trisomy, trisomy 21, trisomy 13,trisomy 14, trisomy 15, trisomy 16, trisomy 18, trisomy 22, triploidytetraploidy, or sex chromosome aneuploidy. In some embodiments, thegenetic abnormality comprises at least one of a trisomy or monosomy. Insome embodiments, labeling comprises labeling the polynucleotide with atleast one of a fluorescent label, a radioactive label, a magnetic label,or a non-optical label. In some embodiments, labeling comprises: nickingone strand of a double-stranded DNA at a first sequence motif with anicking endonuclease; and labeling the DNA. In some embodiments,labeling, further comprises repairing at least some of the nicks on thefirst DNA. In some embodiments, the nicks are not repaired. In someembodiments, the label comprises a transcriptional terminator. In someembodiments, labeling comprises tagging at least one sequence motif ofthe sample molecules with a DNA binding entity selected from the groupconsisting of: a non-cutting restriction enzyme, a zinc finger protein,an antibody, a transcription factor, a transcription activator likedomain, a DNA binding protein, a polyamide, a triple helix formingoligonucleotide, and a peptide nucleic acid, and a methyltransferase. Insome embodiments, labeling with the first label comprises tagging atleast one sequence motif of the sample molecules with amethyltransferase.

According to some embodiments herein, a method of characterizing asample is provided. The method can comprise labeling sample nucleic acidmolecules. The method can comprise translocating the labeled samplenucleic acid molecules through a fluidic nanochannel, wherein thefluidic nanochannel is configured to elongate at least a portion of thesample nucleic acid molecules, and wherein the fluidic nanochannel has alength of at least 10 nm and a cross-sectional diameter of less than1000 nm. The method can comprise detecting signals arising from thesample nucleic acid molecules in the fluidic channels. The method cancomprise determining the positions of the labels on the sample nucleicacid molecules. The method can comprise aligning the positions of thelabels on the sample nucleic acid molecules to the position of labels ina reference genome, wherein the reference genome is obtained from asecond sample from the same organism as the sample molecules.

In some embodiments, the fluidic nanochannel of any of the methodsherein comprises a channel having a length of at least 10 nm and across-section diameter of less than 5000 nm. In some embodiments, thefluidic channel comprises a nanochannel. In some embodiments, thefluidic channel is disposed parallel to a surface of a substrate. Insome embodiments. In some embodiments, the translocating comprisessubjecting the labeled sample to a motivating force selected from thegroup consisting of a fluid flow, a radioactive field, an electroosmoticforce, an electrophoretic force, an electrokinetic force, a temperaturegradient, a surface property gradient, a capillary flow, a pressuregradient, a magnetic field, an electric field, a receding meniscus, asurface tension, a thermal gradient, a pulling force, a pushing force,and a combination thereof.

In some embodiments, the sample of any of the methods herein is selectedfrom the group consisting of a bacteria, a virion, a DNA molecule, anRNA molecule, a nucleic acid polymer, a protein, a peptide, and apolysaccharide. In some embodiments, the sample of any of the methodsherein is derived from maternal blood, and wherein the referencemolecule is derived from a maternal sample other than blood. In someembodiments, the sample of any of the methods herein comprises anucleotide, and wherein the at least two labels are located at eitherend of a zone of interest in the nucleotide. In some embodiments, thesample of any of the methods herein comprises circulating fetal cells,circulating tumor cells, or body fluids or tissues.

In some embodiments, any of the methods herein comprises opticalinspection comprising determining the physical count, the intensity, thewavelength, or the size of the labels. In some embodiments, any of themethods herein comprise optical inspection comprising determining thelength of at least one labeled region in the sample. In someembodiments, any of the methods herein, further comprise determining thesignals arising from a pool comprising the sample or portions of thesample.

In some embodiments, any of the methods herein comprises using the ratio(K) between the signal arising from a plurality of samples or sampleportions (S1, S2 . . . Sn) and the signal arising from the reference(C): K1=S1/C, K2=S2/C . . . Kn=Sn/C In some embodiments, a differencebetween K1 and Kn is used to identify the presence of a fetal sample. Insome embodiments, a difference between K1 and Kn is used to identify thepresence of DNA from a tumor or other cancer source. In someembodiments, a difference between K1 and Kn is used to determine thepresence of a genetic abnormality in the sample. In some embodiments,the genetic abnormality is aneuploidy. In some embodiments, the geneticabnormality is a translocation, addition, amplification, transversion,or inversion.

In some embodiments, any of the methods herein comprises a referencederived from a known diploid or haploid chromosome. In some embodiments,any of the methods herein comprises correlating signals from the samplewith the population distribution from a metagenomic or microbiome study.In some embodiments, any of the methods herein comprises generating ahistogram distribution to reflect coverage depth for the sample.

In some embodiments, a system for characterizing a sample is provided.The system can comprise one or more regions for labeling samplemolecules with at least two labels. The system can comprise a fluidicchannel for translocating the labeled sample molecules, in which thefluidic channel is configured to elongate at least a portion of thesample molecule, and in which the fluidic channel has a length of atleast 10 nm and a cross-sectional diameter of less than 5000 nm. Thesystem can comprise a device for detecting signals arising from thelabeled samples in the fluidic channels.

In some embodiments, a system for characterizing a sample is provided.The system can comprise one or more regions for labeling sample nucleicacid molecules. The system can comprise a fluidic nanochannel fortranslocating the labeled sample nucleic acid molecules, in which thefluidic nanochannel is configured to elongate at least a portion of thesample nucleic acid molecules, and in which the fluidic nanochannel hasa length of at least 10 nm and a cross-sectional diameter of less than1000 nm. The system can comprise a device for detecting signals arisingfrom the sample nucleic acid molecules in the fluidic channels.

In some embodiments a system for characterizing a sample is provided.The system can comprise a region for labeling a plurality ofsequence-specific locations on a sample DNA. The system can comprise aregion for linearizing at least a portion of the sample DNA. The systemcan comprise a device for quantifying the signal arising from the labelson the sample DNA.

In some embodiments, a system for characterizing a sample is provided.The system can comprise a means for labeling sample molecules with atleast two labels. The system can comprise a means for linearizing thelabeled sample molecules. The system can comprise a means for detectingsignals arising from the labeled samples in the fluidic channels.

In some embodiments, a system for characterizing a sample is provided.The system can comprise a means for labeling sample nucleic acidmolecules. The system can comprise a means for linearizing the labeledsample nucleic acid molecules. The system can comprise a means fordetecting signals arising from the sample nucleic acid molecules in thefluidic channels.

In some embodiments, a system for characterizing a sample is provided.The system can comprise a means for labeling a plurality ofsequence-specific locations on a sample DNA. The system can comprise ameans for linearizing at least a portion of the sample DNA. The systemcan comprise a means for quantifying the signal arising from the labelson the sample DNA.

In some embodiments, any of the systems as described herein cancharacterize a sample selected from the group consisting of a bacteria,a virion, a DNA molecule, an RNA molecule, a nucleic acid polymer, aprotein, a peptide, and a polysaccharide. In some embodiments, any ofthe systems as described herein can characterize a sample derived frommaternal blood, and wherein the reference molecule is derived from amaternal sample other than blood. In some embodiments, any of thesystems as described herein can characterize a sample comprising anucleotide, and wherein the at least two labels are located at eitherend of a zone of interest in the nucleotide. In some embodiments, any ofthe systems as described herein can characterize a sample comprisingcirculating fetal cells, circulating tumor cells, or body fluids ortissues.

In some embodiments, any of the systems as described herein can comprisea label selected from the group consisting of a fluorescent label, aradioactive label, a magnetic label, or a combination thereof. In someembodiments, any of the systems as described herein can be configuredfor optical inspection, wherein optical inspection comprises determiningthe physical count, the intensity, the wavelength, or the size of thelabels. In some embodiments, the optical inspection comprisesdetermining the length of at least one labeled region in the sample. Insome embodiments, any of the systems as described herein can beconfigured for correlating the signals, in which correlating the signalscomprises determining the signals arising from a pool of samples or apool of portions of a sample. Some embodiments, any of the systems asdescribed herein can be configured for correlating the signals, in whichcorrelating the signals comprises using the ratio (K) between the signalarising from a plurality of samples or sample portions (S1, S2 . . . Sn)and the signal arising from the reference (C): K1=S1/C, K2=S2/C . . .Kn=Sn/C. In some embodiments, a difference between K1 and Kn is used toidentify the presence of a fetal sample. In some embodiments, adifference between K1 and Kn is used to identify the presence of DNAfrom a tumor or other cancer source. In some embodiments, a differencebetween K1 and Kn is used to determine the presence of a geneticabnormality in the sample. In some embodiments, the genetic abnormalityis aneuploidy. In some embodiments, the genetic abnormality is atranslocation, addition, amplification, transversion, or inversion.

In some embodiments, any of the systems as described herein can comprisea reference derived from a known diploid or haploid chromosome.

In some embodiments, any of the systems as described herein cancorrelated the signals from the sample with the population distributionfrom a metagenomic or microbiome study.

In some embodiments, the fluidic channel any of the systems as describedherein comprises a nanochannel. In some embodiments, the fluidic channelof any of the systems as described herein is disposed parallel to asurface of a substrate. In some embodiments, the translocating comprisessubjecting the labeled sample to a motivating force selected from thegroup consisting of a fluid flow, a radioactive field, an electroosmoticforce, an electrophoretic force, an electrokinetic force, a temperaturegradient, a surface property gradient, a capillary flow, a pressuregradient, a magnetic field, an electric field, a receding meniscus, asurface tension, a thermal gradient, a pulling force, a pushing force,and a combination thereof.

In some embodiments, any of the systems as described herein isconfigured to generate a histogram distribution to reflect coveragedepth for the sample.

In some embodiments a kit for performing any of the methods as describedherein is provided.

In some embodiments a kit for using any of the systems as describedherein is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating sample molecules or particles(ovals) and reference or comparative molecules or particles (spheres)flowing through nanofluidic channels, in accordance with someembodiments herein.

FIG. 2 is a schematic diagram illustrating an embodiment of an imagingsetup to detect signals emitted from labeled molecules or particles totabulate the amount, intensity, and configuration of the sample andreference molecule or particles.

FIGS. 3A-3C are a series of images illustrating small double strandedDNA fragments with known sizes (233 bp, 498 bp, and 834 bp) that weregenerated by PCR, fluorescently stained, flowed, and imaged inindividual nanofluidic channels. FIGS. 3D and 3E show the same doublestranded DNA fragments that were mixed together, flowed, and imaged inthe same nanofluidic channel. The fluorescent signals were plotted in ahistogram (FIG. 3E).

FIGS. 4A-D are a series of graphs illustrating Gaussian curves depictingthe photons emitted from individually labeled DNA molecules with knownsizes (233 bp, 498 bp, and 834 bp). Total counts and intensity werelinearly proportional to mass and/or molecule size. Unknown moleculesizes and quantities can be extrapolated by this method within a lineardynamic range.

FIGS. 5A-5B are a series of scatter plots illustrating the extrapolationof unknown molecule sizes and quantities within a linear dynamic rangeusing the information from FIG. 4. FIGS. 5A-5B show quantitativemeasurement of small fragment molecules or particles: samples of 6different known concentrations of each DNA were prepared; used the sameloading parameters for all the samples of each DNA; performed 3 scansfor each sample; and counted the number of particles in each scan.Initial Results: number of DNAs in the Field of View is linearlyconcentration dependent; the concentration of an unknown DNA of similarsize can be extrapolated and measured; a long range of concentrationscan be covered by changing the loading parameters.

FIG. 6 is a histogram illustrating genomic DNA fragments plotted againsta reference genome (human genome version 19). The y-axis shows coveragedepth for specific chromosomal regions. A uniform distributionthroughout the genome was observed, except for regions without sequenceinformation (such as the centromeres and telomeres).

FIG. 7A is a graph illustrating diploid genomic fragments from a humanmale sample aligned to chromosome 1. The y-axis provides the quantity ofcoverage. The x-axis provides the nucleotide position. The averagecoverage depth was 5X. FIG. 7B is a graph showing a haploid sexchromosome X from the same male sample shown with an average coveragedepth of 2X-2.5X (roughly half of the depth of diploid autosomes),demonstrating the quantitative measurement using the methods andplatform according to some embodiments herein.

DETAILED DESCRIPTION

The fetus sheds small DNA fragments into the maternal bloodstream.Tumors have also been found to release DNA into the bloodstream.According to some embodiments herein are methods for analyzingpolynucleotide fragments such as DNA fragments in blood to detect thepresence of circulating polynucleotide or cells from a fetus or tumor.Also according to some embodiments herein are methods for analyzingfetal DNA in maternal blood to detect genetic abnormalities. In somepreferred embodiments, the methods described herein entail the use of ananofluidic-based single molecule detecting platform to identify geneticabnormalities. Methods and apparatuses in accordance with someembodiments herein have the advantage of analyzing small or largemolecules, such as small or large DNA molecules. In some embodiments, amolecule or region of interest is labeled with at least one pattern, anda reference molecule or region of interest is labeled with at least onepattern. The molecules can be linearized in a microfluidic channel, andcoverage depth for the molecule or region of interest can be compared tocoverage depth for the reference molecule so as to determine copy numberof the molecule of interest.

It is estimated that about 3-15% of short DNAs in maternal blood arefetal derived. Described herein are methods of easily detecting andquantitating small molecules, including short DNA fragments, usingmethods that incorporate fluidics. In some preferred embodiments, themethods comprise quantitating short DNA fragments without sequencing orassembly.

Current prenatal tests involving needle puncture to draw amniotic fluidcan lead to miscarriage and other complications. Further, many currentcancer detection methods also involve invasive procedures, such asbiopsies. According to some embodiments herein, a non-invasive method ofprenatal testing is provided. In some embodiments, the method is fortesting blood. In some embodiments, the method only tests a bloodsample, and does not test a sample from other tissues.

Also described herein are methods of detecting and tracking largermolecules, including longer DNA fragments, to their source using methodsthat incorporate fluidics. For example, in some embodiments, DNAfragments are tracked back to a tumor or other source of cancer. In somepreferred embodiments, the methods are used to track DNA fragments totheir source in order to identify or characterize a genetic abnormality.

In a preferred embodiment, circulating DNA from a maternal blood sampleis analyzed to identify and quantify fetal DNA relative to the maternalgenome. In some embodiments, this information is used to determineprenatal genomic health status (such as trisomy 21) without invasivetests. Examples of suitable oligos for use in an assay for detectinganeuploidy are provided in the HSA21 oligoarray described inYahya-Graison et al., Classification of Human Chromosome 21Gene-Expression Variations in Down Syndrome: Impact on DiseasePhenotypes, Am J Hum Genet 2007, 81(3): 475-491, which is herebyincorporated by reference in its entirety.

In some embodiments, a sample of interest is compared to a referencesample. In some embodiments, the sample of interest is derived from amaternal blood sample. In some of these embodiments, the referencesample is a maternal sample from a source other than blood. In someembodiments, the maternal reference sample includes polynucleotides suchas DNA isolated from a diploid tissue other than blood. In someembodiments, the maternal reference sample comprises a buccal sample, asaliva sample, a urine sample, a sputum sample, or a tear sample. Forexample, in some embodiments, trisomy 21 is detected in a maternal bloodsample compared to a maternal buccal sample.

In some embodiments, the sample of interest is enriched for fetalnucleic acids prior to performing the methods described herein. Forexample, in some embodiments, fetal cells are enriched using a fetalcell specific marker that can be pulled down by an antibody. In someembodiments, the sample of interest undergoes size fractionation.However, any method of enrichment known to one of skill in the art canbe used.

In some embodiments, the sample of interest is derived from a tumor cellor suspected tumor cell, or a tissue in fluid communication with a tumorcell (for example, blood). In some embodiments, the reference sample issample from a healthy cell. In some embodiments, the reference sample isfrom a healthy cell of the same organism as the tumor cell or suspecttumor cell. In some embodiments, the reference sample is selected from atissue that has little to no likelihood of comprising a tumor cell ornucleic acid from the tumor cell.

As one of skill in the art will recognize, the sample of interest mayinclude nucleic acids from a variety of sources. In some embodiments,the sample of interest comprises a bacteria or virion derived from anenvironmental sample, animal or plant tissue, blood, or other bodyfluid. In some embodiments, DNA fragments are used to detect chromosomalabnormalities or cancer genomes.

As one of skill in the art will recognize, the methods described hereincan be used to prepare and analyze DNA from circulating fetal or tumorcells. For example, in some embodiments, cells are lysed to release DNAof interest prior to analysis.

In some embodiments, an entire genome is assayed or analyzed. In someembodiments, only a portion of a genome is assayed or analyzed. In someembodiments, an entire chromosome is assayed or analyzed. In someembodiments, only a portion of a chromosome is assayed or analyzed. Insome embodiments, an entire gene is analyzed. In some embodiments, onlya portion of a gene is assayed or analyzed.

The signals described herein can include any suitable signal, includingoptical signals, fluorescent signals, non-optical signals, radiativesignals, electrical signals, magnetic signals, chemical signals, or anycombination thereof. In some embodiments, signals are generated by anelectron spin resonance molecule, a fluorescent molecule, achemiluminescent molecule, a radioisotope, an enzyme substrate, a biotinmolecule, an avidin molecule, an electrical charged transferringmolecule, a semiconductor nanocrystal, a semiconductor nanoparticle, acolloid gold nanocrystal, a ligand, a microbead, a magnetic bead, aparamagnetic particle, a quantum dot, a chromogenic substrate, anaffinity molecule, a protein, a peptide, a nucleic acid, a carbohydrate,an antigen, a nanowire, a hapten, an antibody, an antibody fragment, alipid, or a combination thereof.

In some embodiments, signals are generated by using one or moreexcitation sources to induce fluorescence, chemoluminescence,phosphorescence, bioluminescence, or any combination thereof. Suitableexcitation sources include lasers, visible light sources, sources ofinfrared light, sources of ultraviolet light, or any combinationthereof.

In some embodiments, the detection of nucleotides or associated signals(for example, fluorophores) is quantitative. In some embodiments, thelength of a nucleotide is quantified. In some embodiments, the size of amolecule is quantified. In some embodiments, the strength of a signalcorrelates with the length of a molecule. For example, as shown in FIG.3A, longer DNA molecules can generate stronger signals than shorter DNAmolecules. In some embodiments, the strength of a signal correlates tothe amount of DNA in a sample or fluidic channel.

In some embodiments, samples are analyzed for copy number variation, forexample, as described in U.S. Patent Publication No. 20130034546, whichis hereby incorporated by reference in its entirety.

The quantity of particular molecules, such as DNA fragments derived fromdifferent chromosomes, can be quantitatively measured in the methodsprovided herein. In some embodiments, the amount of genomic DNA derivedfrom a diploid autosomal chromosome is observed to be twice as much asthat derived from a haploid sex chromosome. In some embodiments, thequantity of such fragments reflects the copy number of a sourcechromosomes. In some embodiments, two or three color labels are used.

In some embodiments, chromosome derived fragments are detected, and arelative ratio is used to identify aneuploidy. In some embodiments, thecopy number of a nucleotide is calculated using the ratios K1=S1/C andK2=S2/C, wherein K1 is the ratio of the signal for a first sample to acontrol sample, and K2 is the ratio of the signal for a second sample tothe control sample. It is contemplated that the copy number from thereference sample is an integer, and that the difference between K1 andK2 can indicate an abnormality in one of the samples of interest. Insome embodiments, the abnormality is detected by comparing the ratio fora particular sample to the average ratio from a plurality of samples.The methods further contemplate that the control genomic sequenceincludes separate portions whose total length per genome is known,wherein the sequence of interest comprises separate portions whoselength per normal gene is known, and wherein a significant differencebetween K1 and K2 indicates a genetic abnormality in the genome. In someembodiments, the nucleotide sequence of interest can relate to atrisomy-linked chromosome, wherein the control genomic sequence is froma chromosome other than the trisomy-linked chromosome, and wherein aK1/K2 ratio of approximately 2:3 or 3:2 indicates a trisomic genotype.In some embodiments, the nucleotide sequence of interest comprises adeletion of a portion of a genome. In some embodiments, the nucleotidesequence of interest comprises a repeating sequence. As such, a copynumber of repeating sequence can be determined according to someembodiments herein. In some embodiments, the first sample comprisesmaternal blood (which, without being limited by any one theory, mayinclude fetal nucleic acids), and the second sample comprises maternaltissue other than blood (preferably a tissue with little to nolikelihood of comprising fetal nucleic acids).

In some embodiments, digital counting detection is performed. In someembodiments, digital counting detection is performed on particles (suchas beads), bacteria, or virion particles. As one of skill in the artwill recognize, the methods described herein can apply to a variety oftargets that can be uniquely labeled. In some embodiments, digitalkaryotyping is performed. For example, in some embodiments, digitalkaryotyping is performed for a chromosome with potential aneuploidy ofinterest. The methods described herein can be used to detect anychromosomal variation of interest, including translocation, addition,amplification, transversion, inversion, aneuploidy, polyploidy,monosomy, trisomy, trisomy 21, trisomy 13, trisomy 14, trisomy 15,trisomy 16, trisomy 18, trisomy 22, triploidy tetraploidy, and sexchromosome abnormalities, including but not limited to XO, XXY, XYY, andXXX.

In some embodiments, methods are provided herein in which the methodsare sensitive enough to detect “short” fragments that are on the orderof tens to hundreds of nucleotides in length. In some embodiments, thesample molecules as described herein comprise polynucleotide “short”fragments. For example, in some embodiments, the polynucleotiodefragments are about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, or 100 nucleotides in length. In some embodiments,the polynucleotide fragments are about 100, 125, 150, 175, 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575,600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925,950, 975, or 1000 nucleotides in length. In some embodiments, themolecules of interest are fragments of less than about 1000, 950, 900,850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200,150, 100, or 50 nucleotides in length. In some embodiments, thefragments are double-stranded. In some embodiments, the fragmentscomprise DNA. In some embodiments, the fragments comprise RNA. In someembodiments, the fragments comprise DNA hybridized to RNA. In someembodiments, the sensitivity is about as high as detecting a singlefluorophore associated with a target fragment.

In some embodiments, the nucleotides of interest are fragments of atleast about 500 nucleotides in length, for example about 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or2000 nucleotides in length, including ranges between any two of thelisted values, for example about 500 to about 2000 nucleotides inlength, about 500 to about 1500, about 500 to about 1000, about 500 toabout 900, about 500 to about 700, about 700 to about 2000, about 700 toabout 1500, about 700 to about 1000, about 700 to about 900, about 1000to about 2000, about 1000 to about 1500, or about 1500 to about 2000.

Molecules suitable for use in the methods and systems described hereininclude polymers, double-stranded DNA, single-stranded DNA, RNA, DNA-RNAhybrids, polypeptides, biological molecules, proteins, and the like.Suitable polymers include homopolymers, copolymers, block copolymers,random copolymers, branched copolymers, dendrimers, or any combinationthereof.

In some embodiments, the methods described herein are sensitive enoughto detect a fetal molecule that constitutes less than about 0.025%,0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%,3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 20%, or 25% of the total number of molecules in amaternal blood sample.

In some embodiments, labeling is directed to a sequence motif orchemical moiety. Labeling can be carried out using any technique knownto one of skill in the art, including chemical or biochemicalconjugation. In some embodiments, the labels described herein are boundto a unique sequence motif. In some embodiments, the labels describedherein are bound to a chemical moiety. In some of these embodiments, thechemical moiety is related to a specific chromosome.

In some embodiments herein, each label is independently selected fromthe group consisting of a fluorophore, a quantum dot, a dendrimer, ananowire, a bead, a hapten, a streptavidin, an avidin, a neutravidin, abiotin, and a reactive group. In some embodiments herein, the first andsecond labels are independently selected from the group consisting of afluorophore or a quantum dot. In some embodiments herein, at least oneof the labels comprises a non-optical label. In some embodiments herein,the labeling is carried out with a polymerase. In some embodimentsherein, the labeling is carried out with a polymerase in the presence ofdNTPs comprising the label. In some embodiments herein, the polymerasehas a 5′ to 3′ exonuclease activity. In some embodiments herein, thepolymerase leaves a flap region, and wherein the flap region is removedto restore a ligatable nick prior to the repairing with a ligase. Insome embodiments herein, the flap region is removed using the 5′ to 3′exonuclease activity of a polymerase under conditions wherein at leastone nucleotide is present in limited concentration. In some embodimentsherein, the flap region is removed using the 5′ to 3′ exonucleaseactivity of a polymerase under conditions wherein at least onenucleotide is omitted from the reaction. In some embodiments herein, theflap region is removed with a flap endonuclease. In some embodimentsherein, the labeling is carried out with a polymerase in the presence ofat least one species of dNTP. In some embodiments herein, the at leastone species of dNTP is a single species of dNTP. In some embodimentsherein, a method as described herein further comprises modulatingactivity of the polymerase by adjusting the temperature, dNTPconcentration, cofactor concentration, buffer concentration, or anycombination thereof, during labeling. In some embodiments herein,nicking the first motif or the second motif comprising nicking withNt.BspQI. In some embodiments herein, the a non-sequence-specific label,for example a polynucleotide backbone label is applied in addition to asequence-specific label or labels as described herein.

In some embodiments, at least one label as described herein comprises anon-optical label. A variety of non-optical labels can be used inconjunction with embodiments herein. In some embodiments a non-opticallabel comprises an electronic label. Exemplary electronic labelsinclude, but are not limited to molecule with a strong electric charge,for example ions such as a metal ions, charged amino acid side chain, orother cations or anions. An electronic label can be detected, forexample, by conductivity (or resistivity) when the label is disposed ina detector. In some embodiments, a nanochannel comprises an electrodeconfigured to determine the presence or absence of an electronic labelby determining the conductivity or resistivity of a substance disposedin the channel. In some embodiments, the non-optical label comprises ametal, metal oxide (for example metal oxide), or silicon oxide moiety.In some embodiments, the non-optical label comprises a moiety (forexample a nanoparticle) comprising a metal, metal oxide, or other oxide.The presence of a particular metal or oxide moiety can be detected, forexample by nuclear magnetic resonance. In some embodiments, the label isconfigured to release a moiety, for example a proton or an anion, upon acertain condition (e.g. change of pH) and the presence or absence ofreleased moiety is detected.

In some embodiments, two or more labels are different from each other.For example, a first motif can be labeled with a first label so as togenerate a first unique pattern, and a second motif that is differentfrom the first motif can be labeled with a second label different fromthe first label so as to generate a second unique pattern. In someembodiments, two or more labels are the same. For example, a first motifcan be labeled with a label, and a second motif that is different fromthe first motif can also be labeled with the same label so as togenerate a unique pattern. In some embodiments, a plurality of probescorresponding to a first chromosome or region of interest are labeledwith a first label, and a second plurality of probes corresponding to asecond chromosome or region of interest (for example a referencechromosome or region) are labeled with a second label that is differentthan the first label. As such, labeled sample molecules comprisingsequences from the first chromosome or region of interest can bedifferentiated from sample molecules comprising sequences from thesecond chromosome or region of interest based on whether they arelabeled with the first label or second label.

Nucleotides with reversible terminators can form a first phosphodiesterlinkage, but prior to reversal of termination, cannot form (or havelimited capacity to form) a second phosphodiester linkage. Thus, anucleotide with a reversible terminator can be incorporated into apolynucleotide (for example at a nick site), but the nucleotide cannotform downstream phosphodiester linkages until the terminator isreversed. Reversal can be performed using techniques known to oneskilled in the art. For example, the terminator can be attached to thenucleotide via cleavable linker, which can be cleaved, for example, viaelectromagnetic radiation. If nick repair is performed using labelednucleotides comprising a 3′ reversible terminator, a single labelednucleotide can be incorporated into the nick, but the terminator canprevent additional labeled nucleotides from being incorporated into thenick. Accordingly, nick labeling can be limited to one labelednucleotide per nick. Limiting nick labeling to one label moiety per nickcan minimize potential bias from multiple labels being incorporated intothe same nick. For example, if approaches are taken to limit labeling toone label moiety per nick, two nicks that are very close together can beresolved based on a relatively strong signal from the label (i.e. thepossibility that two labels simply got incorporated into the same nickcan be ruled-out). For example, if quantitative estimates of the numberof nicks is desired, a one-label-per-nick approach can facilitate directcorrelation between strength of label signal and the number of nicks.The label on the nucleotide comprising a reversible terminator can be asdescribed herein. In some embodiments, the nucleotide comprising areversible terminator comprises a quantum dot. In some embodiments, thenucleotide comprising a reversible terminator comprises a fluorophore.In some embodiments, the nucleotide comprising a reversible terminatorcomprises a non-optical label.

In some embodiments, a plurality of labels label a single samplemolecule. In some embodiments, at least one of the labels comprises asequence specific label. In some embodiments, at least one of the labelscomprises a non-sequence specific label. In some embodiments, at leastone label comprises a sequence specific label, and at least one labelcomprises a non-sequence specific label. In some embodiments, at leastone label does not cut one or both strands of DNA. For example, in someembodiments, at least one label is selected from the group consisting ofa non-cutting restriction enzyme, a methyltransferase, a zinc fingerprotein, an antibody, a transcription factor, a DNA binding protein, ahairpin polyamide, a triplex-forming oligodeoxynucleotide, a peptidenucleic acid, or a combination thereof. In some embodiments, neither thesequence specific nor the non-sequence specific label cuts DNA.

In some embodiments, for example if fluorescent labeling is provided,labeling is detected using a sensitive camera. In some embodiments, forexample if non-optical labeling is provided, labeling is detectedelectronically. However, any detection method can be used that issuitable for the corresponding label. The methods described herein caninclude binding to a fluorescent label, a radioactive label, a magneticlabel, or any combination thereof in one or more regions of themolecules described herein. Binding may be accomplished where the labelis specifically complementary to a molecule or to at least a portion ofa molecule or other region of interest.

In some embodiments, nicking enzymes create sequence-specific nicks thatare subsequently labeled, for example using a labeled nucleotide ornucleotide analog. In some embodiment, the nucleotide or analog isfluorescently labeled. In some embodiments, DNA is linearized byconfinement in a nanochannel, resulting in uniform linearization andallowing precise and accurate measurement of the distance betweennick-labels on DNA molecules comprising a signature pattern. In someembodiments, a second nicking enzyme is used. In some embodiments, thesecond nicking enzyme is used with a second label color. Exemplarynickases that can be used in accordance with embodiments herein include,but are not limited to Nb.BbvCI; Nb.BsmI; Nb.BsrDI; Nb.BtsI; Nt.AlwI;Nt.BbvCI; Nt.BspQI; Nt.BstNBI; Nt.CviPII and combinations thereof.Examples of nicking agents and protocols are also provided in U.S.Patent Application Publication No. 2011/0171634 and U.S. PatentApplication Publication No. 2012/0237936, which are hereby incorporatedby reference in their entireties.

In some embodiments, a polynucleotide, for example an RNA or DNA, islabeled by hybridizing a probe to a single strand of the polynucleotide.The probe can be complementary to a strand of the RNA or DNA or aportion thereof. In some embodiments, the probe is complementary to aparticular sequence motif. In some embodiments, a plurality of probes isprovided so as to be complementary to a plurality of specific sequencemotifs, for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,5,000, or 10,000 probes, including ranges between any two of the listedvalues. In some embodiments, the probe has a random sequence. In someembodiments, a probe with a plurality of random sequences is provided.In some embodiments, a probe includes one or more of an organicfluorophore, quantum dot, dendrimer, nanowires, bead, Au beads,paramagnetic beads, magnetic bead, a radiolabel, polystyrene bead,polyethylene bead, peptide, protein, haptens, antibodies, antigens,streptavidin, avidin, neutravidin, biotin, nucleotide, oligonucleotide,sequence specific binding factors such as engineered restrictionenzymes, methlytransferases, zinc finger binding proteins, and the like.In some embodiments, the probe includes a fluorophore-quencher pair. Oneconfiguration of the probe can include a fluorophore attached to thefirst end of the probe, and an appropriate quencher tethered to thesecond end of the probe. As such, when the probe is unhybridized, thequencher can prevent the fluorophore from fluorescing, while when theprobe is hybridized to a target sequence, the probe is linearized, thusdistancing the quencher from the fluorophore and permitting thefluorophore to fluoresce when excited by an appropriate wavelength ofelectromagnetic radiation. In some embodiments, a first probe includes afirst fluorophore of a FRET pair, and a second probe includes a secondfluorophore of a FRET pair. As such, hybridization of the first probeand the second probe to a single flap, or to a pair of flaps within aFRET radius of each other can permit energy transfer by FRET. In someembodiments, a first probe includes a first fluorophore of a FRET pair,and a label on a nucleotide incorporated to fill a corresponding gap caninclude second fluorophore of a FRET pair. As such, hybridization of thefirst probe to a flap, and the labeled nucleotide into the correspondinggap can permit energy transfer by FRET.

In some embodiments, a double-stranded DNA can be labeled by firstmelting hydrogen bonds between double stands of certain genomic regionsto open a so-called D-loop, by increasing temperature or manipulationwith organic solvent, and then hybridizing to at least one specificprobes with equal or higher affinity to single stranded regions beforeannealing back to relative stable form. As such, in some embodiments,double-stranded DNA can be labeled by a probe as described hereinwithout nicking or cutting either strand. In some embodiments, aplurality of D-loops can be opened on a single strand. As such, aplurality of probes can be annealed to a particular double-stranded DNA.

In some embodiments, labeling comprises transferring a label to thepolynucleotide via a methyltransferase. In some embodiments, themethyltransferase specifically methylates a sequence motif. As such,labeling can comprise transferring a label to a sequence motif by themethyltransferase. Exemplary suitable DNA methyltransferases (MTase)include, but are not limited to, M.BseCI (methylates adenine at N6within the 5′-ATCGAT-3′ sequence), M.Taql (methylates adenine at N6within the 5′-TCGA-3′ sequence) and M.Hhal (methylates the firstcytosine at C5 within the 5′-GCGC-3′ sequence). In some embodiments, twoor more methyltransferases provide two or more labels, which can be thesame or different.

In some embodiments, labeling comprises transferring a label to thepolynucleotide via a methyltransferase. In some embodiments, themethyltransferase specifically methylates a sequence motif. As such,labeling can comprise transferring a label to a sequence motif by themethyltransferase. Exemplary suitable DNA methyltransferases (MTase)include, but are not limited to, M.BseCI (methylates adenine at N6within the 5′-ATCGAT-3′ sequence), M.Taql (methylates adenine at N6within the 5′-TCGA-3′ sequence) and M.Hhal (methylates the firstcytosine at C5 within the 5′-GCGC-3′ sequence). In some embodiments, twoor more methyltransferases provide two or more labels, which can be thesame or different.

In some embodiments, the channel comprises a microchannel. In someembodiments, the channel comprises a nanochannel. Suitable fluidicnanochannel segments have a characteristic cross-sectional dimension ofless than about 1000 nm, less than about 500 nm, or less than about 200nm, or less than about 100 nm, or even less than about 50 nm, about 10nm, about 5 nm, about 2 nm, or even less than about than about 0.5 nm. Afluidic nanochannel segment suitably has a characteristiccross-sectional dimension of less than about twice the radius ofgyration of the molecule. In some embodiments, the nanochannel has acharacteristic cross-sectional dimension of at least about thepersistence length of the molecule. Fluidic nanochannel segmentssuitable for the present invention have a length of at least about 100nm, of at least about 500 nm, of at least about 1000 nm, of at leastabout 2 microns, of at least about 5 microns, of at least about 10microns, of at least about 1 mm, or even of at least about 10 mm.Fluidic nanochannel segments are, in some embodiments, present at adensity of at least 1 fluidic nanochannel segment per cubic centimeter.

Examples of fluidic channels can be found in U.S. Patent Publication No.2008/0242556, which is incorporated herein by reference in its entirety.In some embodiments, a virion particles or a bacterial cell is assayed.For example, in some embodiments, a bacterial cell is assayed using amicrochannel. In some embodiments, the channel allows a cell with adiameter in the range of microns to tens of microns to flow through.

FIG. 1 is a schematic diagram illustrating a fluidic channel arrangementaccording to some embodiments herein. The arrangement can include asample input chamber 10. The arrangement can include an array of fluidicchannels 12, for example fluidic nanochannels. The arrangement caninclude a sample output chamber 14. The output chamber can comprisebuffer solution 16. The array of nanofluidic channels 12 can be in fluidcommunication with the input chamber 10. The array of nanofluidicchannels 12 can be in fluid communication with the output chamber 14.Sample molecules or particles of interest 18 can be disposed in thearray of nanofluidic channels 10. Control or comparative molecules orparticles of interest 18 can be disposed in the array of nanofluidicchannels 10. In some embodiments, the array of nanofluidic channels 12connect the input chamber 10 to the output chamber 14. In someembodiments, sample molecules or particles of interest 18 and control orcomparative molecules or particles of interest 20 are loaded into thesample input chamber, and travel in buffer solution 16 through the arrayof nanofluidic channels. In some embodiments, the sample molecules orparticles of interest 18 and control or comparative molecules orparticles of interest 20 are deposited from the array of nanofluidicchannels 12 into the sample output chamber 14.

FIG. 2 is a schematic diagram illustrating an arrangement for detectionof sample molecules or particles of interest according to someembodiments herein. In some embodiments, the arrangement comprises afirst sample inlet or outlet 11, a second sample inlet or outlet 11, andat least one fluidic channel 13 positioned therebetween and in fluidcommunication with each of the first and second inlet or outlet 11. Itis contemplated herein that if a sample is loaded into the first inletor outlet 11, the first inlet or outlet 11 functions as an inlet and thesecond inlet or outlet 11 can function as an outlet. It is contemplatedherein that if a sample is loaded into the second inlet or outlet 11,the second inlet or outlet 11 functions as an inlet and the first inletor outlet 11 can function as an outlet. In some embodiments, the samplecomprises molecules or particles of interest 18, control or comparativeparticles of interest 20, or a combination of the two. In someembodiments, the molecules or particles of interest 18, control orcomparative particles of interest 20 travel through the fluidic channel13. In some embodiments, the fluidic channel 13 comprises a nanochannel.In some embodiments, the fluidic channel 13 comprises a microchannel. Insome embodiments, the fluidic channel 13 comprises a detection region22. In some embodiments, the system comprises a cover 24 disposed overthe detection region 24. In some embodiments, the cover 24 comprises atransparent cap. In some embodiments, a detector 26 is positioned overthe detection region 22 and the cover 24 (if present). In someembodiments, for example, if optical detection is used, the detector 26comprises a photon detection/imager. In some embodiments, a lens 28 ispositioned in optical communication with the detection region 22 anddetector 26. In some embodiments, the lens 28 is positioned betweendetection region 22 and detector 26. In some embodiments, a dichroicmirror 30 is positioned in an optical communication with the detectionregion 22, lens 28, detector 26, and an excitation source 32, so that afluorescent label, if present, can be excited, and fluorescence from thefluorescent label, if present, can be detected.

In some embodiments, the comparison of samples to a reference sample isprovided in the form of a histogram. In some embodiments, physicalcounts of molecules with a particular labeling pattern that matches to areference or de novo genomic assembly in silico are tabulated in ahistogram distribution to reflect coverage depth. A higher or lower thanaverage coverage depth in specific region or entire chromosome reflectsthe deviation from normal ploidy such as in the case of aneuploidy ingenetic disorder or structural variations in cancer

Additional Alternative Embodiments

Some embodiments described herein can include the following: A method ofcharacterizing a sample, comprising: labeling a region of samplemolecules with at least two labels; translocating the labeled samplemolecules through a fluidic channel, wherein the fluidic channel isconfigured to elongate at least a portion of the sample molecule, andwherein the fluidic channel has a length of at least 10 nm and across-sectional diameter of less than 5000 nm; detecting signals arisingfrom the labeled samples in the fluidic channels; and correlating thesignals arising from the labeled samples to signals arising from thecorresponding region of a reference molecule. The method can furthercomprise: labeling a region of the reference molecule corresponding tothe region of the sample molecules; translocating the labeled referencesample molecule through a fluidic channel, wherein the fluidic channelis configured to elongate at least a portion of the sample molecule, andwherein the fluidic channel has a length of at least 10 nm and across-sectional diameter of less than 5000 nm; and detecting signalsarising from the labeled reference sample in the fluidic channels,wherein the signals arising from a known corresponding region of areference molecule are the signals arising from the labeled referencesample.

In some embodiments, a method of characterizing a sample is provided.The method can comprise: labeling sample nucleic acid molecules;translocating the labeled sample nucleic acid molecules through afluidic nanochannel, wherein the fluidic nanochannel is configured toelongate at least a portion of the sample nucleic acid molecules, andwherein the fluidic nanochannel has a length of at least 10 nm and across-sectional diameter of less than 1000 nm; detecting signals arisingfrom the sample nucleic acid molecules in the fluidic channels;determining the positions of the labels on the sample nucleic acidmolecules; and aligning the positions of the labels on the samplenucleic acid molecules to the position of labels in a reference genome.

In some embodiments, a method of characterizing a sample is provided.The method can comprise: processing double-stranded DNA samples so as togive rise to a flap of the first strand of the double-stranded DNAsamples being displaced from the double-stranded DNA samples, whereinthe flap has a length in the range of from about 1 to about 1000 bases,and wherein the flap gives rise to a gap in the first strand of thedouble-stranded DNA samples corresponding to the flap; incorporating oneor more bases into the double-stranded DNA so as to eliminate at least aportion of the gap; labeling at least a portion of the processeddouble-stranded DNA with one or more tags; and quantifying the signalarising from the labels on the double-stranded DNA; comparing thequantity of the signal arising from the double-stranded DNA to thequantity of the signal arising from a reference DNA; and determining thepresence of a genetic abnormality in the double-stranded DNA when thequantity of the signal arising from the double-stranded DNA differs fromthe quantity of the signal arising from the reference DNA.

In some embodiments, a method of characterizing a sample is provided.The method can comprise labeling a plurality of sequence-specificlocations on a sample DNA; linearizing at least a portion of the sampleDNA; quantifying the signal arising from the labels on the sample DNA;comparing the quantity of the signal arising from the sample DNA to thequantity of the signal arising from a reference DNA; and determining thepresence of a genetic abnormality in the sample DNA when the quantity ofthe signal arising from the sample DNA differs from the quantity of thesignal arising from the reference DNA.

In some embodiments, a system for characterizing a sample is provided.The system can comprise: one or more regions for labeling samplemolecules with at least two labels; a fluidic channel for translocatingthe labeled sample molecules, wherein the fluidic channel is configuredto elongate at least a portion of the sample molecule, and wherein thefluidic channel has a length of at least 10 nm and a cross-sectionaldiameter of less than 5000 nm; and a device for detecting signalsarising from the labeled samples in the fluidic channels.

In some embodiments, a system for characterizing a sample is provided.The system can comprise: one or more regions for labeling sample nucleicacid molecules; a fluidic nanochannel for translocating the labeledsample nucleic acid molecules, wherein the fluidic nanochannel isconfigured to elongate at least a portion of the sample nucleic acidmolecules, and wherein the fluidic nanochannel has a length of at least10 nm and a cross-sectional diameter of less than 1000 nm; and a devicefor detecting signals arising from the sample nucleic acid molecules inthe fluidic channels.

In some embodiments, a system for characterizing a sample is provided.The system can comprise: one or more regions for processingdouble-stranded DNA samples so as to give rise to a flap of the firststrand of the double-stranded DNA samples being displaced from thedouble-stranded DNA samples, wherein the flap has a length in the rangeof from about 1 to about 1000 bases, and wherein the flap gives rise toa gap in the first strand of the double-stranded DNA samplescorresponding to the flap; one or more regions for incorporating one ormore bases into the double-stranded DNA so as to eliminate at least aportion of the gap; one or more regions for labeling at least a portionof the processed double-stranded DNA with one or more tags; and a devicefor quantifying the signal arising from the labels on thedouble-stranded DNA.

In some embodiments, a system for characterizing a sample is provided.The system can comprise: a region for labeling a plurality ofsequence-specific locations on a sample DNA; a region for linearizing atleast a portion of the sample DNA; and a device for quantifying thesignal arising from the labels on the sample DNA.

In some embodiments, a system for characterizing a sample is provided.The system can comprise: means for labeling sample molecules with atleast two labels; means for linearizing the labeled sample molecules;and means for detecting signals arising from the labeled samples in thefluidic channels.

In some embodiments, a system for characterizing a sample is provided.The system can comprise: means for labeling sample nucleic acidmolecules; means for linearizing the labeled sample nucleic acidmolecules; and means for detecting signals arising from the samplenucleic acid molecules in the fluidic channels.

In some embodiments, a system for characterizing a sample is provided.The system can comprise: means for processing double-stranded DNAsamples so as to give rise to a flap of the first strand of thedouble-stranded DNA samples being displaced from the double-stranded DNAsamples, wherein the flap has a length in the range of from about 1 toabout 1000 bases, and wherein the flap gives rise to a gap in the firststrand of the double-stranded DNA samples corresponding to the flap;means for incorporating one or more bases into the double-stranded DNAso as to eliminate at least a portion of the gap; means for labeling atleast a portion of the processed double-stranded DNA with one or moretags; and means for quantifying the signal arising from the labels onthe double-stranded DNA.

In some embodiments, a system for characterizing a sample is provided.The system can comprise: system for characterizing a sample, comprising:means for labeling a plurality of sequence-specific locations on asample DNA; means for linearizing at least a portion of the sample DNA;and means for quantifying the signal arising from the labels on thesample DNA.

According to some embodiments, a method or system as described herein isprovided, wherein the sample is selected from the group consisting of abacteria, a virion, a DNA molecule, an RNA molecule, a nucleic acidpolymer, a protein, a peptide, and a polysaccharide.

According to some embodiments, a method or system as described herein isprovided, wherein the sample is derived from maternal blood, and whereinthe reference molecule is derived from a maternal sample other thanblood.

According to some embodiments, a method or system as described herein isprovided, wherein the sample comprises a nucleotide, and wherein the atleast two labels are located at either end of a zone of interest in thenucleotide.

According to some embodiments, a method or system as described herein isprovided, wherein the label is selected from the group consisting of afluorescent label, a radioactive label, a magnetic label, or acombination thereof.

According to some embodiments, a method or system as described herein isprovided, wherein the optical inspection comprises determining thephysical count, the intensity, the wavelength, or the size of thelabels.

According to some embodiments, a method or system as described herein isprovided, wherein the optical inspection comprises determining thelength of at least one labeled region in the sample.

According to some embodiments, a method or system as described herein isprovided, wherein correlating the signals comprises determining thesignals arising from a pool of samples or a pool of portions of asample.

According to some embodiments, a method or system as described herein isprovided, wherein correlating the signals comprises using the ratio (K)between the signal arising from a plurality of samples or sampleportions (S1, S2 . . . Sn) and the signal arising from the reference(C): K1=S1/C, K2=S2/C . . . Kn=Sn/C. In some embodiments, a differencebetween K1 and Kn is used to identify the presence of a fetal sample. Insome embodiments, a difference between K1 and Kn is used to identify thepresence of DNA from a tumor or other cancer source. In someembodiments, a difference between K1 and Kn is used to determine thepresence of a genetic abnormality in the sample. In some embodiments,the genetic abnormality is aneuploidy. In some embodiments, the geneticabnormality is a translocation, addition, amplification, transversion,or inversion. In some embodiments, the reference is derived from a knowndiploid or haploid chromosome. In some embodiments, the signals from thesample are correlated with the population distribution from ametagenomic or microbiome study.

According to some embodiments, a method or system as described herein isprovided, in which the fluidic channel is a nanochannel. In someembodiments, the fluidic channel is disposed parallel to a surface of asubstrate. In some embodiments,

According to some embodiments, a method or system as described herein isprovided, further comprising generating a histogram distribution toreflect coverage depth for the sample.

According to some embodiments, a method or system as described herein isprovided, wherein the sample comprises circulating fetal cells,circulating tumor cells, or body fluids or tissues.

According to some embodiments, a method or system as described herein isprovided, wherein the translocating comprises subjecting the labeledsample to a motivating force selected from the group consisting of afluid flow, a radioactive field, an electroosmotic force, anelectrophoretic force, an electrokinetic force, a temperature gradient,a surface property gradient, a capillary flow, a pressure gradient, amagnetic field, an electric field, a receding meniscus, a surfacetension, a thermal gradient, a pulling force, a pushing force, and acombination thereof.

According to some embodiments, a kit for performing a method asdescribed herein is provided.

According to some embodiments, a kit for using the system of any one ofthe preceding claims is provided.

In the description provided herein, reference is made to theaccompanying drawings, which form a part hereof. The illustrativeembodiments described in the detailed description, drawings, and claimsare not meant to be limiting. Other embodiments may be utilized, andother changes may be made, without departing from the spirit or scope ofthe subject matter presented here. It will be readily understood thatthe aspects of the present disclosure, as generally described herein,and illustrated in the Figures, can be arranged, substituted, combined,and designed in a wide variety of different configurations, all of whichare explicitly contemplated and make part of this disclosure.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

As used herein, the term “channel” means a region defined by borders.Such borders may be physical, electrical, chemical, magnetic, and thelike. The term “nanochannel” is used to clarify that certain channelsare considered nanoscale in certain dimensions.

As used herein, the term “DNA” refers to DNA of any length (e.g., 0.1 Kbto 1 megabase). The DNA can be a highly pure preparation, crude, or semicrude material. The DNA can come from any biological source or can besynthetic.

As used herein, the term “nucleotide” refers to a molecule containingdeoxyribonucleic acids (e.g., DNA, mtDNA, gDNA, or cDNA), ribonucleicacid (e.g., RNA or mRNA), or any other variant of nucleic acids known inthe art. The term “labeled nucleotide” refers to a nucleotide comprisingany modification that is detectable. This includes but is not limited tonucleotides with reporter groups attached to the base. Reporter groupsinclude but are not limited to fluorescent dyes, haptens, biotinmolecules or gold nanoparticles. The term “native nucleotide” refers toa nucleotide that is not modified, or has a slight modification thatdoes not interfere with its incorporation into DNA. The terms “t”, “c”,“a”, “g” and “u” refer to nucleotides in DNA.

The term “nick” refers to a phosphodiester bond break occurring on oneDNA strand or the other, having a 3′ hydroxyl end.

As used herein, the term “nicking endonuclease” refers to any enzyme,naturally occurring or engineered, that is capable of breaking aphosphodiester bond on a single DNA strand leaving a 3′-hydroxylat adefined sequence. Nicking endonucleases can be naturally occurring,engineered by modifying restriction enzymes to eliminate one DNA strandcutting activity, or produced by fusing a nicking subunit to a DNAbinding domain, for example, zinc fingers and transcription activatorlike effectors DNA recognition domains.

As used herein, the term “labeling sites” refers to any DNA site with anexposed 3′ hydroxyl group onto which the polymerase can add nucleotidesin a template dependent manner. Labeling sites can be generated bynicking endonucleases, hybridized probes, or any chemical or physicalmeans of breaking a phosphodiester bond on any one DNA strand. Means ofbreaking a phosphodiester bond can occur to DNA outside its biologicalsource or prior to DNA extraction, for example as a result of abiological sample exposure to chemicals, and external forces such asradiation. If 3′ ends are not extendable, repair can be performed torestore the hydroxyl group, for example by using New England Biolabs'PreCR kit.

As used herein a “sample” can include, for example, blood, serum,plasma, sputum, lavage fluid, cerebrospinal fluid, urine, semen, sweat,tears, saliva, and the like. As used herein, the terms “blood,” “plasma”and “serum” expressly encompass fractions or processed portions thereof.Similarly, where a sample is taken from a biopsy, swab, smear, etc., the“sample” expressly encompasses a processed fraction or portion derivedfrom the biopsy, swab, smear, etc.

As used herein, the term “chromosome” refers to the heredity-bearinggene carrier of a living cell which is derived from chromatin and whichcomprises DNA and protein components (especially histones).

As one of skill in the art will recognize, “translocating” can be usedinterchangeably with linearizing when used in the context passing a DNAmolecule through a nanochannel.

The methods, apparatuses, systems, and kits described herein canincorporate the methods, apparatuses, systems, and kits described in anyof the following references: U.S. Patent Application Publication No.2009/0305273; PCT Publication No. WO/2008/079169; U.S. PatentApplication Publication No. 2008/0242556; PCT Publication No.WO/2008/121828; U.S. Patent Application Publication No. 2011/0171634;PCT Publication No. WO/2010/002883; U.S. Patent Application PublicationNo. 2011/0296903; PCT Publication No. WO/2009/149362; U.S. PatentApplication Publication No. 2011/0306504; PCT Publication No.WO/2010/059731; U.S. Patent Application Publication No. 2012/0097835;PCT Publication No. WO/2010/135323; PCT Application No. PCT/US11/57115;U.S. patent application Ser. No. 13/606,819; PCT Application No.PCT/US2012/054299; U.S. Patent Application Publication No. 2012/0244635;PCT Publication No. WO/2011/038327; U.S. Patent Application PublicationNo. 2012/0237936; U.S. patent application Ser. No. 13/503,307; PCTPublication No. WO/2011/050147; U.S. Patent Application Ser. No.61/734,327; U.S. Patent Application Ser. No. 61/761,189; and U.S. PatentApplication Ser. No. 61/713,862, which are each hereby incorporated byreference in their entireties.

Example 1

Genomic fragments from a human male sample were generated by PCR,labeled, and run through a nanochannel. Detected fragments were thenaligned to a single gene reference optical map for each chromosome. Themolecules were sorted based on the alignment start site.

As shown in FIG. 7A, the average coverage depth observed for a diploidautosomal chromosome (chromosome 1) was 5X, and was evenly distributedacross the chromosome. If the sampling of molecules had been even, thealignment start sites would have been randomly distributed across thechromosome, resulting in a linear plot.

As shown in FIG. 7B, the average coverage depth observed for a haploidsex chromosome (chromosome X) from the same male sample was 2X-2.5X(roughly half the depth of diploid autosomes), and was also evenlydistributed across the chromosome. This example demonstrates thequantitative measurements that can be achieved using the methods andplatform described herein.

1.-84. (canceled)
 85. A method of characterizing a sample comprisingpolynucleotide sequences, the method comprising: labeling a plurality ofsequence-specific locations on a polynucleotide sequence of a samplemolecule of less than 550 nucleotides in length; linearizing at least aportion of the sample molecule in a fluidic channel; quantifying asignal from the labels on the sample molecule; comparing a quantity ofthe signal from the sample molecule to a quantity of signal from areference molecule of less than 550 nucleotides in length, so as tocompare coverage depth of polynucleotide sequences of the samplemolecule to coverage depth of polynucleotide sequences of the referencemolecule; and determining a presence or absence of a genetic abnormalityin the sample molecule when the coverage depth of the sample moleculediffers from the coverage depth of the reference molecule.
 86. Themethod of claim 85, wherein the sample comprises circulating fetalcells.
 87. The method of claim 85, further comprising generating ahistogram distribution to reflect coverage depth for the sample.
 88. Themethod of claim 85, wherein the sample molecule and the referencemolecule are from different tissues of the same organism.
 89. The methodof claim 85, wherein the sample molecule is derived from maternal blood,and wherein the reference molecule is derived from a maternal sampleother than blood.
 90. The method of claim 85, wherein the quantity ofsignal from the reference molecule comprises an electronically oroptically stored value or set of values.
 91. The method of claim 85,wherein the genetic abnormality comprises at least one of atranslocation, addition, amplification, transversion, inversion,aneuploidy, polyploidy, monosomy, trisomy, trisomy 21, trisomy 13,trisomy 14, trisomy 15, trisomy 16, trisomy 18, trisomy 22, triploidy,tetraploidy, and sex chromosome aneuploidy.
 92. A system forcharacterizing a sample, comprising: one or more regions for labelingsample nucleic acid molecules of less than 550 nucleotides in length; afluidic nanochannel for translocating the labeled sample nucleic acidmolecules of less than 550 nucleotides in length, wherein the fluidicnanochannel is configured to elongate at least a portion of the samplenucleic acid molecules; and a device configured to detect physicalcounts of signals arising from the labeled sample nucleic acid moleculesof less than 550 nucleotides in length in the fluidic channels.
 93. Thesystem of claim 92, wherein the sample comprises circulating fetalcells, circulating tumor cells, or body fluids or tissues.
 94. Thesystem of claim 92, wherein the system is configured to correlatesignals arising from the labeled sample nucleic acid molecules tosignals arising from the corresponding region of a reference molecule.95. The system of claim 93, wherein the sample is derived from maternalblood, and wherein the reference molecule is derived from a maternalsample other than blood.
 96. The system of claim 93, wherein the signalsarising from a known corresponding region of a reference molecule arethe signals arising from a labeled reference molecule.
 97. The system ofclaim 93, wherein correlating the signals comprises determining thesignals arising from a pool of samples or a pool of portions of asample.
 98. The system of claim 93, wherein the system is configured tocorrelate the signals comprises using the ratio (K) between the signalarising from a plurality of samples or sample portions (S1, S2 . . . Sn)and the signal arising from the reference (C):K1=S1/C,K2=S2/C . . . Kn=Sn/C
 99. The system of claim 98, wherein adifference between K1 and Kn is used to determine the presence of agenetic abnormality in the sample.
 100. The system of claim 99, whereinthe genetic abnormality comprises at least one of a translocation,addition, amplification, transversion, inversion, aneuploidy,polyploidy, monosomy, trisomy, trisomy 21, trisomy 13, trisomy 14,trisomy 15, trisomy 16, trisomy 18, trisomy 22, triploidy, tetraploidy,sex chromosome aneuploidy, or any combination thereof.
 101. The systemof claim 92, wherein the system is configured to generate a histogramdistribution to reflect coverage depth for the sample.
 102. The systemof claim 92, wherein the labeling comprises labeling the nucleic acidmolecules with at least two labels located at either end of a zone ofinterest in the nucleic acid molecule.
 103. The system of claim 92,wherein the detecting comprises optical inspection comprisingdetermining the physical count, the intensity, the wavelength, or thesize of the labels, and wherein the detecting comprises opticalinspection comprising determining the length of at least one labeledregion in the sample.
 104. The system of claim 92, wherein the fluidicnanochannel has a length of at least 10 nm and a cross-sectionaldiameter of less than 1000 nm.